The severity of radiation injury in humans is largely determined by high sensitivity of the gastrointestinal (GI) tract, yet no drugs are approved for this indication. This proposal is focused on a novel clinical-stage drug candidate, Protectan CBLB502, a toll-like receptor 5 (TLR5) agonist capable of effective mitigation of otherwise lethal radiation-induced GI injury in mice and non-human primates (NHPs). A comprehensive understanding of mechanisms of action is required for drug development under the FDA Animal Efficacy Rule. Accordingly, the main objective of this proposal is to use mouse and NHP models to characterize in detail the mitigating effect of CBLB502 on various elements of the GI system and to identify cellular and molecular mediators of this effect. Specifically, our multi-institutional collaborative team will: (i) create a comprehensive "histological atlas" illustrating the mitigating effects of CBLB502 on various elements of GI infrastructure damaged by radiation, (ii) define the target organs that contribute to the GI radiomitigation activity of CBLB502 with specific focus on bone marrow and liver, and (iii) identify primary and secondary cellular and molecular responders to CBLB502 with the expectation of defining molecular conductors of the radiomitigating function of the drug as well as novel efficacy biomarkers. Completion of this program should create a solid mechanistic base for translation of animal data into a well-justified projected human efficacious dose and will be critical for FDA approval of CBLB502 as a medical countermeasure to mitigate GI radiation damage.